U.S. patent number 6,777,000 [Application Number 09/795,897] was granted by the patent office on 2004-08-17 for in-situ gel formation of pectin.
This patent grant is currently assigned to Carrington Laboratories, Inc.. Invention is credited to Yawei Ni, Kenneth M. Yates.
United States Patent |
6,777,000 |
Ni , et al. |
August 17, 2004 |
**Please see images for:
( Certificate of Correction ) ** |
In-situ gel formation of pectin
Abstract
In-situ gelation of a pectic substance. Composition, method of
preparation, and method of use of a pectin in-situ gelling
formulation for the delivery and sustained release of a
physiologically active agent to the body of an animal. The pectin
can be isolated from Aloe vera.
Inventors: |
Ni; Yawei (College Station,
TX), Yates; Kenneth M. (Grand Prairie, TX) |
Assignee: |
Carrington Laboratories, Inc.
(Irving, TX)
|
Family
ID: |
25166729 |
Appl.
No.: |
09/795,897 |
Filed: |
February 28, 2001 |
Current U.S.
Class: |
424/488; 514/1;
514/157; 514/2.4; 514/44R; 514/19.3 |
Current CPC
Class: |
A61P
31/04 (20180101); A61K 47/36 (20130101); A61K
9/0024 (20130101); A61P 17/02 (20180101) |
Current International
Class: |
A61K
47/36 (20060101); A61K 9/00 (20060101); A01K
009/14 (); A01N 061/00 (); A01N 043/04 () |
Field of
Search: |
;514/1,2,44,157
;424/488 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 98/47535 |
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Apr 1998 |
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WO |
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WO 98/47535 |
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Oct 1998 |
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WO |
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WO 99/27905 |
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Jun 1999 |
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WO |
|
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|
Primary Examiner: Nguyen; Dave T.
Assistant Examiner: Schnizer; Richard
Attorney, Agent or Firm: Needle & Rosenberg, P.C.
Claims
What is claimed is:
1. A method for sustained release of a physiologically active agent
to an animal, comprising: a) providing a liquid solution or
dispersion comprising i) a liquid carrier, ii) a pectic substance
having a degree of methylation of less than 30% and an average
molecular weight of greater than 4.6.times.10.sup.5 Daltons, in an
amount effective to gel the liquid solution or dispersion when
applied to the tissues or body fluids of the animal, and iii) one
or more physiologically active agents; and b) applying the liquid
solution or dispersion to the tissues or body fluids of the animal
to form a gel comprising one or more physiologically active agents
in contact with the tissues or body fluids.
2. The method of claim 1 wherein the pectic substance has a degree
of methylation of less than about 10%.
3. The method of claim 2 wherein the pectic substance has an
average molecular weight of greater than about 0.785.times.10.sup.6
Daltons.
4. The method of claim 2 wherein the pectic substance has an
average molecular weight of greater than about 1.times.10.sup.6
Daltons.
5. The method of claim 4 wherein the pectic substance has a
galacturonic acid content of greater than about 90% w/w.
6. The method of claim 4 wherein the pectic substance comprises
rhamnose at greater than about 4% by mole.
7. The method of claim 4 wherein the pectic substance comprises
3-methoxy-rhamnose.
8. The method of claim 4 wherein the pectic substance is an aloe
pectin.
9. The method of claim 4 wherein the pectic substance comprises
from about 0.1% to about 20% of the total weight of the solution or
dispersion.
10. The method of claim 4 wherein the pectic substance comprises
from about 0.25% to about 2% of the total weight of the solution or
dispersion.
11. The method of claim 4 wherein the pectic substance comprises
about 0.5% w/v of the solution or dispersion.
12. The method of claim 4 wherein the pectic substance is
amidated.
13. The method of claim 1 wherein the solution or dispersion
comprises a sodium salt.
14. The method of claim 1 wherein the solution or dispersion is
capable of monovalent cation-based gel formation at 4.degree.
C.
15. The method of claim 14 wherein the monovalent cation is
sodium.
16. The method of claim 4 wherein the solution or dispersion
comprises a thickener.
17. The method of claim 4, wherein the solution or dispersion
comprises carboxyrnethylcellulose, hydroxypropylmethylcettular
gelatin, dextran, hyaluronic acid, or alginate.
18. The method of claim 4, wherein the carrier comprises water or
saline.
19. The method of claim 1 wherein the solution or dispersion is not
administered to a mucosal surface.
20. The method of claim 19 wherein the solution or dispersion is
administered topically.
21. The method of claim 1 wherein the solution or dispersion is
administered to a wound.
22. The method of claim 1 wherein the solution or dispersion is
administered to a surgical site.
23. The method of claim 1 wherein the solution or dispersion is
administered subcutaneously.
24. The method of claim 1 wherein the solution or dispersion is
administered intraperitoneally.
25. The method of claim wherein the solution or dispersion is
administered parenterally.
26. The method of claim 1 wherein the solution or dispersion is
administered to an organ.
27. The method of claim 1 wherein the solution or dispersion is
administered into a tumor.
28. The method of claim 1 wherein the solution or dispersion is
administered to a joint cavity.
29. The method of claim 4 wherein the physiologically active agent
comprises a pharmacologically active substance, a therapeutic
agent, a diagnostic agent, a peptide, a nucleic acid, or a
protein.
30. The method of claim 4 wherein the physiologically active agent
is a protein.
31. The method of claim 4 wherein the physiologically active agent
is a peptide.
32. The method of claim 4 wherein the physiologically active agent
is a vaccine.
33. The method of claim 4 wherein the physiologically active agent
is a nucleic acid.
34. The method of claim 4 wherein the physiologically active agent
is a diagnostic agent.
35. The method of claim 4 wherein the gel provides a sustained
release of the physiologically active agent to the tissues or
bodily fluids.
36. The method of claim 1 that does not require application of a
component comprising cross-linking ions.
37. The method of claim 1 wherein the pectic substance has been
filtered to provide a solution which is clearer than that obtained
without filtration of the pectic substance.
38. The method of claim 4 wherein the pectic substance has been
filtered to provide a solution which is clearer than that obtained
without filtration of the pectic substance.
39. The method of claim 4 wherein the physiologically active agent
is an anti-bacterial agent.
40. The method of claim 4 wherein the physiologically active agent
is silver sulfadiazine.
41. The method of claim 4 wherein the solution or dispersion is
administered to a wound.
42. The method of claim 4 wherein the solution or dispersion is
administered to a surgical site.
43. The method of claim 4 wherein the solution or dispersion is
administered subcutaneously.
44. The method of claim 4 wherein the solution or dispersion is
administered intraperitoneally.
45. The method of claim 4 wherein the solution or dispersion is
administered parentally.
46. The method of claim wherein the solution or dispersion is
administered to an organ.
47. The method of claim 4 wherein the solution or dispersion is
administered into a tumor.
Description
BACKGROUND
The present invention relates to in-situ gelation of a pectic
substance. Specifically, the invention relates to a pectin in-situ
gelling formulation for the delivery and sustained release of a
physiologically active agent to the body of an animal. More
specifically, the pectic substance is derived from Aloe vera L.
plant.
Abbreviations Used Herein Include:
CMC, carboxylmethyl cellulose; Da, dalton; DM, degree of
methylation; Gal A, galacturonic acid; HEC, hydroxyethyl cellulose;
HM, high methoxyl; HPMC, hydroxypropylmethylcellulose; kDa,
kilodaltons; LM, low methoxyl; PBS, phosphate buffered saline;
PEG-PLGA-PEG, polyethylene glycol-poly(lactic-co-glycolic
acid)-polyethylene glycol; PEO-PLLA, poly(ethylene
oxide)-poly(L-lactide); PEO-PPO-PEO, poly(ethylene
oxide)-poly(propylene oxide)-poly(ethylene oxide).
Pectin is a biodegradable acidic carbohydrate polymer. Pectin is
commonly found in plant cell walls. The cell wall of a plant is
divided into three layers consisting of the middle lamella, the
primary wall and the secondary cell wall. The middle lamella is
richest in pectin. The chemistry and biology of pectin have been
extensively reviewed (Pilnik and Voragen, Advances in plant
biochemistry and biotechnology 1, 219-270, 1992; Voragen et al, In
Food polysaccharides and their applications. pp 287-339. Marcel
Dekker, Inc. New York, 1995; Schols and Voragen, In Progress in
Biotechnology 14. Pectins and pectinases, J. Visser and A. G. J.
Voragen (eds.). pp. 3-20. Elsevier Science Publishers B. V.
Amsterdam, 1996).
Pectin consists of an .alpha.-(1.fwdarw.4)-linked polygalacturonic
acid backbone intervened by rhamnose residues and modified with
neutral sugar side chains and non-sugar components such as methyl
and acetyl groups. The extent of rhamnose insertions and other
modifications vary depending on plant sources. The Gal A content is
generally more than 70% whereas the rhamnose content is typically
<2%. Rhamnose residues are .alpha.-(1.fwdarw.2)-linked to Gal A
residues in the backbone. They cause the formation of a T-shaped
kink in the backbone chain, and the increase in rhamnose content
leads to more flexible molecules. The neutral sugar side chains are
attached to the rhamnose residues in the backbone at the O-3 or O-4
position. The rhamnose residues tend to cluster together on the
backbone. Hence, this region with side chains attached is referred
to as the "hairy region" while the rest of the backbone is named
the "smooth region."
Methylation occurs at carboxyl groups of Gal A residues. The degree
of methylation or methyl-esterification ("DM") is defined as the
percentage of carboxyl groups (Gal A residues) esterified with
methanol. Based on the DM, pectins are divided into two classes,
low methoxyl ("LM") pectin with a DM of <50% and a high methoxyl
("HM") pectin with a DM of >50%. Commercial pectins derived from
citrus and apples are naturally HM pectins. LM pectins are
typically obtained through a chemical de-esterification process.
Commercial LM pectins typically have a DM of 20-50%. A completely
de-esterified pectin is referred as "pectic acid" or
"polygalacturonic acid". Pectic acid in the acid form is insoluble
but is soluble in the salt form. The common salt form of pectic
acid is either sodium or potassium.
Pectin is most stable at acidic pH levels between approximately
3-4. Below pH 3, methoxyl and acetyl groups and neutral sugar side
chains are removed. Under neutral and alkaline conditions, methyl
ester groups are saponified and the polygalacturonan backbone
breaks through .beta.-elimination-cleavage of glycosidic bonds on
the non-reducing ends of methylated Gal A residues. Pectic acids
and LM pectins are relatively more resistant to neutral and
alkaline conditions since there are only limited numbers of methyl
ester groups or none at all.
Current commercial pectins are mainly from citrus and apples.
However, besides citrus and apples, pectins can also be isolated
from many other plants. All vegetables and fruits that have been
examined contain pectins. Pectins from sugar beets, sunflowers,
potatoes, and grapefruits are just a few other well known
examples.
Both HM and LM pectins form gels. However, these gels form via
totally different mechanisms (Voragen et al, In Food
polysaccharides and their applications. pp 287-339. Marcel Dekker,
Inc. New York, 1995). HM pectin forms a gel in the presence of high
concentrations of co-solutes (sucrose) at low pH. LM pectin forms a
gel in the presence of calcium, thus, it is "calcium-reactive." The
calcium-LM pectin gel network is built by formation of what is
commonly referred to as an "egg-box" junction zone in which Ca++
causes the cross-linking of two stretches of polygalacturonic acid
chains.
HM pectins are generally not reactive with calcium ions and
therefore cannot form a calcium gel. However, certain HM pectins
have been reported to be calcium sensitive and capable of calcium
gel formation. In addition, HM pectins can be made calcium-reactive
by a block wise de-esterification process while still having a DM
of >50%. See, Christensen et al. U.S. Pat. No. 6,083,540.
Calcium-LM pectin gel formation is influenced by several factors,
including DM, ionic strength, pH, and molecular weight (Garnier et
al., Carbohydrate Research 240, 219-232, 1993; 256, 71-81, 1994).
The lower the DM and the higher the molecular weight, the more
efficient the gelation. Furthermore, the calcium-LM pectin gelation
is more efficient at a neutral pH of .about.7.0 than .about.3.5.
Lastly, the addition of monovalent counter ion (NaCl) enhances the
gelation, i.e., less calcium is required for gel formation.
Pectins are typically utilized in the food industry and classified
by the FDA as "GRAS" (Generally Regarded As Safe). They have also
long been used as colloidal and anti-diarrhea agents. Recently,
pectins have been utilized in the areas of medical device and drug
delivery (Thakur et al., Critical Reviews in Food Science &
Nutrition 37, 47-73, 1997). In the case of drug delivery, pectin
has found its presence in many experimental formulations for oral
drug delivery to the colon because pectin is readily degraded by
bacteria present in this region of the intestines. The pectin is
either used directly with no gelation involved or a pectin calcium
gel is preformed to encapsulate the drug agent before
administration. Ashford et al., J. Controlled Release 26, 213-220,
1993; 30, 225-232, 1994; Munjeri et al., J. Controlled Release 46,
273-278, 1997; Wakerly et al., J. Pharmacy & Pharmacology 49,
622-625, 1997; International Journal of Pharmaceutics
153,219-224,1997; Miyazaki et al., International Journal of
Pharmaceutics 204, 127-132, 2000. Prior to the present invention,
there appears to be no attempt made to examine the in-situ gelling
ability of pectins.
Aloe pectin isolated from Aloe vera plant as described in U.S. Pat.
No. 5,929,051, the entire content of which is incorporated herein
by reference. It is naturally a LM pectin and capable of calcium
gelation. In addition, it possesses several unique chemical
properties that are particularly related to gelation, including a
high molecular weight (>1.times.10.sup.6 Da), a high Gal A
content (as high as >90%), and a low DM (<10%).
Current commercial pectins typically have a size of
7-14.times.10.sup.4 Da and Gal A content of .about.75% (Voragen et
al, In Food polysaccharides and their applications. pp 287-339.
Marcel Dekker, Inc. New York, 1995). These pectins have a rhamnose
content of <2%. Commercial LM pectins and other natural LM
pectins have a DM of >20%. A DM below 10% makes Aloe pectin
nearly a pectic acid. A pectin with such a low DM, a high molecular
weight, and a high Gal A content has not been described previously.
Aloe pectin is an off white powder as the finished product, whereas
all current commercial and experimental pectins are yellow to tan
powders.
Drug delivery has been a subject of intense studies over recent
years. The goal is to achieve sustained (or slow) and/or controlled
drug release and thereby improve efficacy, safety, and/or patient
comfort. A sustained and/or controlled release of the drug agents
is achieved by the retardation of drug diffusion by and/or gradual
disintegration of the polymer matrix following application.
In-situ gelation is a process of gel formation at the site of
application after the composition or formulation has been applied
to the site. In the field of human and animal medicine, the sites
of application refers to various injection sites, topical
application sites, surgical sites, and others where the agents are
brought into contact with tissues or body fluids. As a drug
delivery agent, the in-situ gel has an advantage related to the gel
or polymer network being formed in-situ providing sustained release
of the drug agent. At the same time, it permits the drug to be
delivered in a liquid form.
Polymers capable of in-situ gelation have been described. They
include Poloxamer, Pluronics (Vadnere et al., Int. J. Pharm., 22,
207-218, 1984), various copolymers such as PEO-PLLA and
PEG-PLGA-PEG (Jeong et al., Nature 388, 860-862, 1997; Jeong et
al., J. Controlled Release 63, 155-163, 2000), cellulose
acetophalate latex (Gurny et al. J. Controlled Release 353-361,
1985), Gelrite (Rozier et al., Int. J. Pham. 57, 163-168, 1989),
Carbopol, and Matrigel. The gel formation is induced by temperature
change (Poloxamer, Pluronics, PEO-PLLA diblock copolymer,
PEG-PLGA-PEG triblock copolymer, and Matrigel), pH change
(cellulose acetophalate latex and Carbopol), or reaction with mono-
or di-valent cations (Gelrite). However, most of them require a
high polymer concentration for in-situ gel formation (>20%)
(Poloxamer, PEO-PLLA diblock copoly, PEG-PLGA-PEG triblock
copolymer, cellulose and acetophalate latex). The thermally gelling
polymers (Poloxamer, Pluronics, PEO-PLLA diblock copolymer,
PEG-PLGA-PEG triblock copolymer, and Matrigel) also have the
disadvantage of gelling before administration due to temperature
change during packaging or storage. Unfortunately some of these
polymers are not biodegradable such as Poloxamer or require
manipulation of the temperature before administration (PEO-PLLA
diblock copolymer) or during formulation (Pluronics and Gelrite).
An ophthalmic in-situ gelling drug delivery formulation consisting
of a mixture of Carbopol and Pluronic was found to be more
effective than formulations consisting of either one. However,
Pluronic is used at 14% (Lin and Sung, Journal of Controlled
Release 69, 379-388, 2000). Such polymers are therefore not well
suited for medical applications in humans and animals. Furthermore,
many of these polymers form only a hydrogel which is a viscous but
still flowing solution (e.g., Poloxamer and Pluronics).
The in-situ gelation compositions using ionic polysaccharides have
been disclosed in U.S. Pat. No. 5,958,443, which consist of a drug,
a polymer and a gel forming ionic polysaccharide which consist of
two components, an ionic polysaccharide and a cross-linking ion
capable of cross-linking the former. The in-situ gel formation is
induced by the application of the cross-linking ions.
Thus, a great need exists for a simpler and more efficient in-situ
gelling composition that employs only a low polymer concentration
for the purposes of drug delivery.
SUMMARY OF THE INVENTION
One embodiment of the present invention pertains to using a pectic
substance to provide a biodegradable in-situ gelling composition
for animal and human use. The composition transforms from a liquid
into a gel following administration to the target site. Preferably
the pectic substance is Aloe pectin.
One object of the present invention is to provide a composition for
controlled, or sustained, release of a physiologically active agent
in the body of an animal.
Another object of the present invention is to provide for a
transparent polymer solution wherein no dramatic increase in gel
cloudiness is created beyond certain concentration ranges.
Preferably the composition is capable of creating an in-situ gel at
low concentrations.
Another object of the present invention is to provide for a
transparent polymer solution wherein a thickener is added.
Preferably the composition is capable of creating an in-situ gel at
low concentrations.
A further object of the present invention is to provide for a
composition that is capable of creating an in-situ gel at low
concentrations once delivered in the liquid form.
Another object of the present invention is to provide for a
composition for drug delivery. In the case of drug delivery, for
example, a therapeutic or diagnostic agent is incorporated into the
formulation or composition. These agents can be small molecules as
well as large ones such as proteins. Preferably the composition is
capable of forming an in-situ gel at low concentrations.
These and other objects of the present invention are provided by
the described embodiments of the present invention. The foregoing
discussion has outlined some of the more pertinent features of the
present invention. These should be construed to be merely
illustrative of some of the more prominent features and
applications of the invention. Accordingly, a fuller understanding
of the invention maybe had by referring to the following Detailed
Description.
BRIEF DESCRIPTION OF THE DRAWINGS
For a more complete understanding of the preferred embodiment of
the present invention, reference is made to the following detailed
description taken in conjunction with the accompanying drawings,
wherein like numerals refer to like elements, wherein:
FIG. 1 is a bar graph representing the relationship of NaCl to the
calcium gelation of Aloe pectin.
FIG. 2 shows the Aloe pectin in-situ gelation at various Aloe
pectin concentrations with normal animal serum.
FIG. 3 shows the Aloe pectin in-situ gelation in the presence of a
thickener (HEC or alginate) with normal animal serum.
FIG. 4 shows the slow release effect obtained with Aloe pectin
in-situ gel using a small organic compound (fast green).
FIG. 5 shows a bar graph representing the relationship between bFGF
treatment and cell number in a defined area.
DETAILED DESCRIPTION
Thus, the general term "pectic substance," as used in this
invention, includes pectin, low and high methoxyl pectin,
de-esterified pectin, pectin calcium gel, Aloe pectin sodium gel,
pectic acid, pectate, pectinic acid, pectinate, protopectin, and
pectin-rich substances, such as Aloe vera inner gel cell wall
fiber, individually, collectively, or in combination thereof. As
discussed above, pectin is a group designation for those complex
colloidal carbohydrate derivatives which occur in, or are prepared
from, plants and contain a large proportion of anhydrogalacturonic
acid units which are thought to exist in a chain-like combination.
The carboxyl groups may be partially esterified by methyl groups
and partially or completely neutralized by one or more bases. Thus,
"deesterified" usually means that one or more methyl ester groups
have been removed from the pectin molecules. "Pectic acids" is the
group designation applied to pectic substances mostly composed of
colloidal polygalacturonic acids and essentially free from methyl
ester groups. The totally de-esterified pectin is pectic acid or
polygalacturonic acid. "Pectates" are either normal or acid salts
of pectic acids. "Pectinic acids" are the colloidal
polygalacturonic acids containing more than a negligible proportion
of methyl ester groups. "Pectinates" are either normal or acid
salts of pectinic acids. "Protopectin" is applied to the
water-insoluble parent pectin which occurs in plants and which upon
restricted hydrolysis yields pectins, pectinic acids, and others.
The water-insoluble pectin may be associated with the cellulose
present in the plant, such as the Aloe vera inner gel or rind cell
wall fiber.
Aloe Pectin
Aloe vera leaves consist of two parts, an outer green rind and a
clear inner gel which is also referred to as pulp. Aloe pectin is
extracted from the inner gel or outer rind cell wall fibers. Use of
a chelating agent at a slight alkaline pH is found to be the most
efficient extraction method. Aloe pectin is unique as compared to
previously described pectins. It has a high rhamnose content of
>4% in the purified pectin preparation which is at least 2 times
higher than described in other pectins such as citrus, apple, sugar
beet, and sunflower. Rhamnose is a key sugar in the pectin backbone
whose content affects the flexibility of the molecule. Aloe pectin
also possesses a rare sugar, 3-OMe-rhamnose which has not been
described in any other pectins. Aloe pectin is naturally LM, having
a DM generally <30% and can be as low as <10%. The Gal A
content of Aloe pectin is >70% and can be as high as >90%.
Aloe pectin is capable of gel formation in the presence of calcium.
A monovalent cation, such as sodium, potassium and lithium
accelerates the formation of gel.
Aloe pectin can be distinguished from other pectins by one or more
of the following characteristics:
1) A high molecular weight (>1.times.10.sup.6 Da) and a high
intrinsic viscosity (>550 ml/g);
2) A high rhanmose content (>4%);
3) A high galacturonic acid content (>90%);
4) Containing 3-OMe-rhamnose;
5) Being naturally LM with a DM as low as <10%;
6) Capable of calcium gel formation;
7) Capable of monovalent cation-based gel formation at low
temperature (4.degree. C.).
We found that by injecting into a body or by topically applying to
wound surfaces as a route of administration, a non-gelled liquid
pectin can form a gel in-situ at the site of administration. The
in-situ gel is firm and non-flowing just like the calcium gel
formed in vitro, which is distinct from the hydrogel, a viscous but
still flowing solution. The in-situ gelation of Aloe pectin was
found to be particularly efficient such that the minimal Aloe
pectin concentration needed for forming a firm solid in-situ gel is
as low as 2.5 mg/ml or 0.25% (w/v) and can be even lower if a
thickener is added.
The gel compositions can be made isotonic or iso-osmotic and
adjusted to the pH of mammalian body fluids, such as lacrimal
tears. The pH and osmotic pressure of such bodily fluids are 7.4
and 29 mOsm/kg, respectively. It is advantageous to deliver a
pharmacologically active medicament to an area of the mammalian
body requiring pharmacological treatment under desired pH and
osmotic pressure conditions which, for instance, match those of
bodily fluids. Optionally, the pharmaceutical compositions of the
invention can be provided in a sterile condition.
Although not wanting to be bound by any theory, it is believed that
the pectin in-situ gelation is primarily mediated by the calcium
ions in the body fluids. Blood has a calcium concentration of
8.5-10.3 mEq/dl. The calcium gelation of pectins is enhanced in the
presence of NaCl which is also a normal component of the body
fluids. There are 134 mEq/L NaCl in the blood.
The in-situ gel also forms in the presence of various agents,
including small organic compounds, proteins, nucleic acid, live
cells, and other polymers following subcutaneous injection,
demonstrating the capability of the pectin for delivering a wide
range of agents in an encapsulated or entrapped form. When a poorly
soluble compound such as silvadene was incorporated, the in-situ
gel still formed. Once delivered, the pectin in-situ gel clearly
exerted a slow release effect. This was demonstrated under in vitro
as well as in vivo conditions with a small organic model compound
(fast green). In addition, when bFGF is delivered with the pectin
in-situ gel, a significantly increased cell proliferation
surrounding the gel was observed.
Aloe pectin is more efficient than current commercial pectins
including LM pectins, and polygalacturonic acid, and amidated LM
pectins for in-situ gelation. A well-formed in-situ gel was only
obtained with commercial polygalacturonic acid or LM pectin at a
concentration 10 times higher than that for Aloe pectin. Current
commercial LM pectins and polygalacturonic acids have a lower Gal A
content (.about.75%), a much lower molecular weight
(7-14.times.10.sup.4 Da), and a DM of 20-50%. There are other
polymers that can form a calcium gel. One example is alginate.
However, alginate was not capable of forming a well defined in-situ
gel at concentrations tested. Alginate is a polysaccharide block
copolymer consisting of guluronic acid (G) and manuronic acid (M)
(Moe et al., In Food polysaccharides and their applications. pp
287-339. Marcel Dekker, Inc. New York, 1995). These two residues in
alginates exist as G-block, M-block, or alternating MG-block. Only
the G-block is responsible for calcium gelation. The total G
content varies widely dependent on the sources; the highest G
content is .about.70%. In addition, the alginate calcium gelation
is inhibited by the presence of NaCl, which exists in the
physiological fluids.
Several other polymers have also been shown to be capable of
in-situ gelation. However, most of them require a high polymer
concentration for in-situ gel formation (>20%) (Poloxamer,
PEO-PLLA diblock copoly, PEG-PLGA-PEG triblock copolymer, cellulose
and acetophalate latex). Some of these polymers are not
biodegradable, such as Poloxamer, or require manipulation of the
temperature before administration (PEO-PLLA diblock copolymer) or
during formulation (Pluronics and Gelrite). The thermally gelling
polymers (Poloxamer, Pluronics, PEO-PLLA diblock copolymer,
PEG-PLGA-PEG triblock copolymer, and Matrigel) also have the
disadvantage of gelling before administration due to ambient
temperature changes during packaging or storage. Furthermore, many
of these polymers form only a hydrogel, a viscous but still flowing
solution (e.g., Poloxamer and Pluronics). In addition, some polymer
formulations require two different polymers or the application of a
second component for gelation to occur.
Pectin, especially the Aloe pectin, is advantageous over these
polymers or compositions in that the polymer concentration required
to achieve the in-situ gelation is very low (.gtoreq.0.25%, w/v)
and can be even lower if a thickener is added. The preparation does
not require temperature or pH adjustment, or application of a
second component for the in-situ gelation to occur. The gel is
transparent, and there is no dramatic increase in gel cloudiness
beyond certain concentration ranges as with PEG-PLGA-PEG triblock
copolymer and Pluronics.
The advancement of biotechnology is generating more and more
protein-based therapeutics. Proteins are inherently unstable.
Proper formulation and delivery are critical to their in vivo
functions (Langer, Nature 392, 5-10, 1998; Putney and Burke, Nature
Biotechnology 16, 153-157, 1998). The pectin in-situ gel is
particularly suited for protein delivery because of its mild
gelling conditions. Many protein agents are also intended to be
delivered locally in a sustained manner, e.g., growth factors for
wound healing and angiogenic factors for therapeutic angiogenesis.
This can also be achieved with pectin in-situ gel. When bFGF was
delivered with the Aloe pectin in-situ gel, a significantly
increased cell proliferation surrounding the gel was observed.
As used herein, the term "physiologically active agent" refers to
an agent that can exert a physiological response in the body of an
animal. The physiologically active agent includes, for example, a
pharmacologically active substance; a small molecule, such as an
inorganic compound, an organic compound and its salt thereof; a
diagnostic agent; a therapeutic agent; a nucleic acid; a peptide; a
polymer; a small protein; a large protein; and a live cell. A
pharmacologically active substance includes a substance that
illicits immune response, such as a vaccine. Examples of
therapeutic agents include anti-bacterial substances, antimicrobial
agents, antiparasitic agents, antibiotics, antihistamines,
decongestants, antimetabolites, antiglaucoma agents, anti-cancer
agents, antiviral agents, anti-fungal agents, anti-inflammatory
agents, anti-diabetic agents, anesthetic agents, anti-depressant
agents, analgesics, anti-coagulants, opthalmic agents, angiogenic
factors, immunosuppressants, and anti-allergic agents. Based on the
weight of the final composition or formulation, the physiologically
active agent can vary from about 0.01% to about over 90%. The
amount of the physiologically active agent used would depend on the
type, form, and nature of the physiologically active agent.
The range of the pectic substance can vary from about 0.01% to
about 40%, based on the total weight of the composition, preferably
from about 0.1% to about 20%, and more preferably from about 0.25%
to about 2%. The amount of the pectic substance used would depend
on the type, form, and nature of the physiologically active agent.
Optionally, a carrier or excipient may be used.
A carrier used for this invention includes any pharmaceutically
acceptable carrier, such as water; saline; a buffered aqueous
solution; emulsion, such as oil/water emulsion; adjuvant; a wetting
agent; tablet; and capsule. Based on the weight of the final
composition or formulation, the carrier can vary from about 0% to
about 90%. The amount of carrier present would depend on the
physiologically active agent and the manner by which the
formulation or composition is to be delivered.
Representative buffering agents include alkali or alkali earth
carbonate, chloride, sulfate, phosphate, bicarbonate, citrate,
borate, acetate, and succinate. Representative preservatives
include sodium bisulfite, sodium thiosulfate, ascorbate,
benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric
borate, paraben, benzylalcohol, and phenylethanol.
Thus, one embodiment of the current invention is to provide a
composition for the sustained delivery of a physiologically active
compound, and the composition contains a pectin and a
physiologically active compound with or without a pharmaceutically
acceptable thickener. Preferably, the composition changes from a
liquid to a gel upon administration of the composition to the body
of an animal, and thus the release of the physiologically active
compound is sustained or controlled.
A biodegradable thickener such as CMC, HPMC, sodium alginate,
collagen, gelatin, and hyaluronic acid may be added to the
formulation. Addition of such a thickener does not influence the
gelling efficiency as described below, but provides an advantage of
enhancing the density of the gel matrix and the in-situ gel
formation at lower pectin concentrations. In addition, polymers
that are responsive to changes in pH, ionic strength, and
temperature may also be used as long as they are synergistic with
the pectin gelation. Furthermore, a blend of different pectins may
be used with or without a thickener. Other thickeners include
Carbopol, Gelrite, chitosan, and xyloglucan. Based on the weight of
the final composition or formulation, the thickener can vary from
about 0% to about 90%. The amount of biodegradable thickener used
would depend on the physiologically active agent and the manner of
which the composition or formulation is used.
Still another embodiment of the current invention is to provide a
composition consisting of a pectin with or without a
pharmaceutically acceptable thickener for use as a medical
device.
Preferably, the pectic substance is a calcium reactive. More
preferably, the pectic substance is a LM pectin or polygalacturonic
acid. Still more preferably, the pectic substance is Aloe
pectin.
The calcium reactivity can be determined by methods including gel
formation, change in viscosity, and potentiometry.
The term "gel formation" refers to an increase in viscosity of a
solution triggered by a change in the physical or chemical
condition. The gel formed can be a viscous liquid, a solid, or in
any state in between. Gels in various states may be obtained by
adjusting the polymer concentration or other factors. A gel in a
particular state can be most suited for certain applications.
A pectin in-situ gelling compositions containing a therapeutic or
diagnostic agent(s) may be administered or delivered to the animal
by various means. For example, it may be applied topically to the
eyes, mucosal surfaces, or wounds. It may also be delivered
parenterally, such as subcutaneously, intramuscularly, or via
intraperitoneal injection. It may also be injected into an organ, a
joint cavity, or a tumor.
Pectin can be extracted from many different plant sources. Besides
citrus and apples, for example, pectin has also been obtained from
potatoes, grape fruits, sugar beets, and sunflower heads. Pectin
may be modified. For example, an amidated pectin is produced by
treatment with ammonia. It is conceivable that an Aloe-pectin-like
pectin may be present in a different plant species or a pectin from
a different plant source may be produced, re-processed, and/or
modified in a way to enhance the in-situ gelling ability based on
the principles disclosed herein. Furthermore, although an LM pectin
with a DM >50% is preferred for use in the present invention
because of its calcium reactivity, certain HM pectins are also
known to be calcium-sensitive and capable of forming calcium gel,
and may therefore be used for in-situ gelling (Tibbits et al.,
Carbohydrate research 310, 101-107, 1998). In addition, a block
wise de-esterified HM pectin that still has a DM of >50%, but is
rendered calcium sensitive by the block wise de-esterification, may
also be used. See, Christensen et al. U.S. Pat. No. 6,083,540.
Thus, it should be appreciated by those skilled in the art that the
specific embodiments disclosed above may be readily utilized as a
basis for modifying or designing other structures for carrying out
the same purposes of the present invention. It should also be
realized by those skilled in the art that such equivalent
constructions do not depart from the spirit and scope of the
invention as set forth in the appended claims.
EXAMPLE 1
In-situ Gelation of Aloe Pectin
Extraction of Aloe pectin
Aloe pectin was extracted from cell wall fibers prepared from
either pulp or rind of Aloe vera leaves. The general methods of
extracting pectins have been reported. See, Voragen et al, In Food
polysaccharides and their applications. pp 287-339. Marcel Dekker,
Inc. New York, 1995. See also, U.S. Pat. No. 5,929,051, the entire
content of which is hereby specifically incorporated by reference.
The extraction of Aloe pectin was achieved with a chelating agent
such as EDTA or under other conditions including hot water, hot
diluted acid (HCl, pH 1.5-3), and cold diluted base (NaOH and
Na.sub.2 CO.sub.3 ; pH 10).
Following the extraction, the remaining fibers were removed by
coarse and fine filtrations. The pectin was precipitated with
ethanol. The pectin precipitates were further rinsed with ethanol
solutions before being dried.
Aloe pectins obtained in this manner from either pulp or rind cell
wall fibers were characterized with a molecular weight
(.gtoreq.1.times.10.sup.5 Da), a low DM (<50%), and a Gal A
content (>80%). Preferably, the molecular weight was
>1.times.10.sup.6 Da, the DM was <10%, and the Gal A content
was >90%.
The molecular weight was determined by HPLC-based size exclusion
chromatography with pullulan as the standard. DM was determined by
a selective reduction method (Maness et al., Analytical
Biochemistry 185, 346-352, 1990) and a HPLC-based method (Voragen
et al., Food Hydrocolloids, 1, 65-70, 1986). Gal A content was
determined by m-hydroxyldiphenyl method (Blumenkrantz, N. and
Asboe-Hansen, G. Analytical Biochemistry 54, 484-489, 1973). The
content of each of these three references is hereby incorporated by
reference.
In-situ gelation of Aloe pectin
Aloe pectin was first dissolved in sterile deionized water and then
mixed with equal volume of 2.times.physiological saline (0.3 M
NaCl). Aloe pectin could not be readily dissolved in salt solution.
However, once dissolved in water, the pectin can be mixed with the
salt solution to achieve the physiological ionic strength. The
pectin solution in physiological saline obtained in this manner
remained clear. The pectin solutions were free-flowing at room
temperature and had a pH of 5.0-6.0 depending on the polymer
concentrations. No adjustment of temperature or pH was performed
unless otherwise indicated. The preparation was injected
subcutaneously into lower abdominal regions of Swiss Webster mice
(0.05 or 0.1 ml per site) in accordance with the animal use
protocols. Mice were sacrificed at various times following
injection and the gel formation was examined.
The swelling of the skin at the injection site did not disappear
over time as in the case of the saline control. When the skin over
the injection site was surgically incised, a piece of gel shaped
like a ball or an oval was observed. The gel was clear,
transparent, and firm. It could be readily separated from
surrounding tissues. The gel was surgically excised along with
skin, fixed in formalin, sectioned, stained with H&E, and
examined under the microscope. The gel was only lightly stained but
was clearly visible and surrounded by the dermal tissues. The same
in-situ gelation was also observed in rats. The swelling at the
injection site was not as evident in rats as in mice due to the
thicker skin and hair coat. However, when skin at the injection
site was surgically incised, the same in-situ gel was observed.
With rats, one ml of Aloe pectin solution could be injected
subcutaneously at the lower abdominal region and correspondingly
much larger gel pieces were obtained.
The gel formation is pectin concentration-dependent. At a
concentration of .gtoreq.0.25% (w/v), a solid firm gel was
obtained. No gel formation was observed at .ltoreq.0.1% (w/v). At
concentrations between 0.1% and 0.25%, a soft gel was obtained.
The in-situ gel also formed when the pH of the Aloe pectin solution
was adjusted to .about.7.2 with dilute sodium hydroxide.
The in-situ gelling ability is dependent on the molecular weight of
Aloe pectin. When an Aloe pectin with a much reduced molecular
weight (.about.3.times.10.sup.4 Da) but the same DM and Gal A
content was used, no in-situ gelation was observed when tested at
0.5% (w/v).
The in-situ gel also formed following injection through
intraperitoneal and intramuscular routes although the gel formed
did not appear to have as uniform a shape as that formed following
subcutaneous injection.
EXAMPLE 2
In-situ Gelation Following Topical Application to Wound Surface
Aloe pectin preparation (0.5%, w/v) in physiological saline was
directly applied to fresh full-thickness excisional skin wounds on
mice or rats. A 0.5% (w/v) CMC preparation in physiological saline
and a commercial hydrogel wound dressing were used as a control.
The wounds were made with a biopsy punch in accordance with animal
use protocols. After 4 hrs, rats were sacrificed and wounds
surgically removed. Wounds were fixed in formalin, sectioned, and
stained with H&E. A layer of gel was clearly formed on the
surface of wounds with the Aloe pectin preparation but not with CMC
or the commercial hydrogel wound dressing.
EXAMPLE 3
Pectin In-Situ Gelation Mediated by Calcium Ions
Body fluids such as blood and lacrimal fluid contain calcium ions
(8.5-10.3 mEq/dl in blood). Since Aloe pectin forms calcium gel,
the role of calcium in the in-situ gelation of Aloe pectin was
examined using an in vitro gelling system with animal serum that
mimics the in-situ gel formation. This in vitro assay is described
as a gel frontal migration assay in which animal serum was placed
at the bottom of a glass tube and the Aloe pectin solution is
layered on top of the serum (the pectin solution may be placed at
the bottom of the tube dependent on the density of the test
solution in relation to the pectin solution). The gel formed in the
pectin phase can be distinguished from the pectin solution by its
increased turbidity when examined under a light source. Also,
tilting the tube does not move the interface if a gel is
formed.
Tissue culture grade normal calf serum was used. Two ml of serum
was placed at the bottom of a glass tube (0.8.times.11 cm) and 1 ml
pectin solution (0.5-0.75%, w/v) was placed on top of it. The gel
formation was immediate at the contact line or interphase and the
gel phase or gel front gradually extended upward in the pectin
solution over time. However, if the serum was first dialyzed
against saline or EDTA (a chelator for divalent cations) or EGTA (a
specific chelator for calcium) was added to the serum to a final
concentration of 10 mM, no gel formation was observed. This
indicates that the calcium is responsible for the pectin in-situ
gelation.
The pectin in-situ gelation also occurred with heparinized whole
mouse blood or plasma isolated therefrom.
EXAMPLE 4
Pectin In-Situ Gelation with Other Body Fluids
Besides serum or blood, there are many other types of body fluids
such as tear fluid. To determine if the pectin in-situ gelation
also occurred with other body fluids, the gel frontal migration
assay described in Example 3 was used along with Aloe pectin (0.25%
in saline).
The gel formation occurred with the peritoneal fluid. In this case,
the ascites from mice injected with hybridoma for monoclonal
antibody production was used as peritoneal fluid.
The gel formation also occurred with simulated body fluids. They
are:
1) Tear fluid (0.68 g NaCl, 0.22 g NaHCO.sub.3, 0.008 g
CaCl.sub.2.2H.sub.2 O, and 0.14 g KCl per 100 ml. (See,
Stjernschantz and Asitin, in Edman, P. (ed.), "Biopharmaceutics of
Ocular Drug Delivery," CRC Press, Boca Raton, pp. 1-15, 1993.
Alternatively, 0.268 g bovine serum albumin, 0.268 glysozyrne,
0.134 g globulin, 0.008 g CaCl.sub.2.2H.sub.2 O, 0.650 g D-glucose,
and 0.658 g NaCl per 100 ml. See, Cohen et al., Journal of
Controlled Release 44, 201-208, 1997);
2) Lung fluid (0.01 g MgCl.sub.2.6H.sub.2 O, 0.61 g NaCl, 0.03 g
KCl, 0.027 g Na.sub.2 HPO.sub.4.7H.sub.2 O, 0.007 g Na.sub.2
SO.sub.4, 0.018 g CaCl.sub.2.2H.sub.2 O, 0.095 g
NaHC.sub.2.3H.sub.2 O, 0.26 g NaHCO.sub.3, and 0.01 g Na.sub.3 H
C.sub.6 O.sub.7.2H.sub.2 O per 100 ml. See, Fisher and Briant,
Radiation Protection Dosimetry, 53, 263-267, 1994); and
3) Nasal secretion (0.867 g NaCl, 0.44 g Na.sub.2 HPO.sub.4, 0.108
g NaH.sub.2 PO.sub.4, 0.058 g CaCl.sub.2.2H.sub.2 O, 0.31 g KCl
0.636 g albumin per 100 ml. See, Lorin et al., Journal of
Laboratory Clinical Medicine, 2, 275-267, 1994).
EXAMPLE 5
NaCl Enhances Pectin Calcium Gelation
Body fluids such as blood and lacrimal fluids also contain sodium
ions (135-146 mEq/L in blood). Pharmacological preparations for
topical or parenteral use are prepared in a buffered or
non-buffered physiological saline (0.15 M NaCl) or isotonic
solution. NaCl has been shown to enhance the calcium gelation of LM
pectins. To determine if the same effect occurs with Aloe pectin,
the same gel frontal migration assay was used. Aloe pectin (0.5%,
w/v) solutions prepared in 0.15 M NaCl (2 ml) were placed at the
bottom of the tube and a 100 mM CaCl.sub.2 solution (0.05 ml) was
placed on top of the pectin solution. The gel formed extending
downward in the pectin solution over time. The migration of the gel
front was measured at 18 hrs following the addition of CaCl.sub.2.
The results showed that the gel front migrated faster in the
presence of NaCl, i.e., the calcium gelation of Aloe pectin was
enhanced by the presence of NaCl (FIG. 1). The effect of NaCl was
also dose-dependent; the gel formation rate was faster in 0.15 M
NaCl than in 0.05 M NaCl.
These observations are consistent with previous findings with other
LM pectins (Gamier et al., Carbohydrate Research 240, 219-232,
1993; 256, 71-81, 1994). FIG. 1 is a bar graph representing the
relationship of NaCl to the calcium gelation of Aloe pectin.
EXAMPLE 6
Pectin In-Situ Gelation is Faster at Low Pectin Concentrations
The gel frontal migration assay described above was used. Aloe
pectin at various concentrations in saline (1 ml) was applied onto
the normal calf serum (2 ml). After 18 hrs at room temperature, the
length of gels formed was measured. The initial gelation at the
contact phase is immediate regardless of the pectin concentration.
However, the rate at which the gel length grew over time differed
at different pectin concentrations. It was found that the lower the
pectin concentration, the faster the gelation; the length of the
gel formed at 0.05% (w/v) was nearly 5 times longer than that at
0.5% (w/v) (FIG. 2). The gel formed at low concentrations
(<0.2%, w/v) was much softer and could be broken by strong
agitation.
The same observation was also made when a calcium chloride solution
was used to replace the serum. This indicates that the rate of
pectin calcium gelation is increased at lower pectin
concentrations.
EXAMPLE 7
Addition of Other Polymers or Thickeners Enhances the Pectin
In-Situ Gel Formation
The gel frontal migration assay described above was used. Polymers
such as HEC (0.45%, w/v), CMC (0.45%, w/v), or sodium alginate
(0.45%, w/v) were mixed with Aloe pectin (0.05%, w/v). Sodium
alginate, although capable forming calcium gel with CaCl.sub.2
solutions under in vitro conditions, did not form an in-situ gel
with the serum. One ml of the polymer solution was applied onto 2
ml normal calf serum. The length of gels formed was measured 18 hrs
later. The results showed that addition of other polymers did not
influence the rate of the pectin in-situ gelation (FIG. 3). The
same result was also obtained when the polymer was mixed with Aloe
pectin at a different ratio (0.4% vs 0.1%).
In the in vivo mouse model, a mixture of Aloe pectin (0.375%, w/v)
and CMC (0.375%, w/v) in saline formed an in-situ gel following
subcutaneous injection. In addition, the addition of a thickener
(sodium alginate or HEC at 0.4% or 0.3%, w/v) made it possible to
obtain a better formed in-situ gel at low Aloe pectin
concentrations (0.1% or 0.2%, w/v) at which the in-situ gels were
either soft or not formed with Aloe pectin alone (Example 1).
EXAMPLE 8
Comparison with Other Pectins and Alginates
LM pectins that are capable of calcium gelation were used in the
experiments. They included a LM pectin from citrus with a DM of 28%
and a polygalacturonic acid prepared from apple pectin (DM=0), both
of which were obtained from Sigma Chemical Co., and an amidated
pectin with a DM of 28-34% and a DA (degree of amidation) of
16-22%. Before use, they were dissolved in de-ionized water,
filtered, ethanol precipitated, and dried.
The in-situ gelation experiment in mouse by subcutaneous route was
performed as described in Example 1. Four injection sites on two
mice were used for each sample. The results showed that following
subcutaneous injection, no in-situ gel formation was observed with
any of them at a concentration of 1.0 or 1.65% (w/v). Only
smear-like gel substances were observed. However, when tested at a
higher concentration (3.0 or 3.3%, w/v), well formed gels were
obtained with both polygalacturonic acid and amidated LM
pectin.
Similarly, the low molecular weight of Aloe pectin described in
Example 1 also gelled in situ at a high concentration (2.5%,
w/v).
An HM citrus pectin with a DM of 64% was also tested. It was
prepared in the same way as that for the LM pectins. No gel
formation was observed for the HM pectin at a concentration of 3%
(w/v). The injection site was wet and watery and no solid gel
pieces were observed.
Alginates were also tested, including Keltone HVCR and the high G
alginate Manugel DMB (G content, 60-70%) at a concentration of
0.5%. Only a smear-like gel substance was observed when examined 4
hrs post subcutaneous injection, indicating that most of the
materials had diffused away without gelling. The alginates also did
not form a gel with the normal animal serum in the in vitro in-situ
gelation assay as described above (Example 7).
These results together showed that the LM pectin, polygalacturonic
acid, amidated LM pectin, and alginate are much less efficient than
Aloe pectin for in-situ gelation, under the same
concentrations.
EXAMPLE 9
Delivery of Physiologically Active Agents by Pectin In-Situ Gel
For the in-situ gelation to be used for drug delivery, the
phenomena must occur in the presence of the drug or diagnostic
agents. Thus, various compounds or agents were mixed with Aloe
pectin in physiological saline with a final pectin concentration of
0.5% (w/v). These compounds or agents were a small organic compound
(fast green, 808 Da, 10 mg/ml), a small protein (bFGF, 17 kDa, 10
.mu.g/ml), a medium-sized protein (bovine serum albumin, 66 kDa, 10
mg/ml), a large-size protein (type I bovine collagen, 2 mg/ml), a
nucleic acid (Lamda DNA Hind III fragments, 200 .mu.g/ml), a
carbohydrate polymer (CMC, 0.5%, w/v), and Raw 264.7 cells (a mouse
macrophage line, 1.times.10.sup.8 /ml). The mixtures were injected
subcutaneously into mice. Gel formation was then examined 4 hrs
after injection. The results showed that the gel formation occurred
in the presence of all the agents tested as the Aloe pectin alone
control. The concentrations of the drug agents used were those
tested and were not the maximum concentration possible.
Furthermore, by gel frontal migration assay, the in-situ gelation
of a 0.5% (w/v) Aloe pectin solution also occurred in the presence
of 1) 0.1% (w/v) silvadene (silver sufadiazine), a poorly soluble
anti-bacteria agent commonly used for wound treatment, 2) 0.5%
(w/v) hydroxyethyl cellulose (HEC), and 3) 0.5% (w/v) sodium
alginate (Keltone HVCR, Kelco). The presence of 0.5% (w/v) HEC or
sodium alginate did not influence the efficiency of the in-situ
gelation as described in Example 6.
Thus, the fact that the in-situ gelation occurred with these many
different agents clearly indicates that the pectin in-situ gel can
be used for delivery of a wide range of drug agents.
EXAMPLE 10
Slow Release of a Small Organic Compound from Pectin In-Situ Gels
Under
In Vitro Condition
Therapeutic and diagnostic agents vary greatly in molecular weight,
from .about.100 Da to over 10,000 Da. Generally, the smaller the
compound, the more difficult to achieve a slow release effect. Here
a small organic compound was chosen as a test model. It is a dye,
fast green, which has a molecular weight of 808 and is widely used
in the food and pharmaceutical industry. The dye was mixed with
Aloe pectin (0.5%, w/v) in saline at a concentration of 1 mg/ml. A
1 mg/ml dye solution in saline only was used as a control. One ml
of the dye/pectin preparation or the control was placed into a
dialysis tube (1 cm in diameter) with a 12 kDa cut-off. Dialysis
tubes with samples were then placed into 25 ml normal calf serum in
30-ml glass tubes. One serum tube receiving the dye/Aloe pectin
solution also received EDTA to a final concentration of 10 mM to
prevent calcium gelation. The serum tubes containing the samples
were then shaken continuously at 100 rpm on a rotatory shaker. A
small amount of serum (100 .mu.l) was sampled at various time
points. The amount of dye released into the serum was determined by
measuring the OD at 620 nm. Serum samples with known amounts of
fast green were used to establish the standard curve. The results
showed that similar amounts of fast green were released from the
control and dye/Aloe pectin with EDTA (without gel formation) and
the amount of the dye released from the dye/Aloe pectin without
EDTA (with gel formation) was significantly lower (p<0.05;
student t-test) over the time points measured (FIG. 4). This
indicates that the presence of Aloe pectin and its gelation
significantly slowed the release of the compound.
EXAMPLE 11
Slow Release of a Small Organic Compound from Pectin In-Situ Gels
following
Subcutaneous Injection
To determine if the above observed slow release could be obtained
under in vivo conditions, the fast green (1 mg/ml)/Aloe pectin
(0.5%, w/v) in physiological saline or fast green in physiological
saline alone was injected subcutaneously into mice. The injection
sites (two per sample) were examined 4 hrs later. It was found that
with the presence of pectin, in-situ gels were formed, which
retained the dye although the color was not as strong as the
original preparation prior to injection. In contrast, the injection
sites of the control had no color and thus no retained dye.
Therefore, the pectin in-situ gel retained the dye and indeed
slowed the release under the in vivo condition.
EXAMPLE 12
Local Delivery of bFGF by Aloe Pectin In-Situ Gel
For growth factors to exert local effect on tissues surrounding the
administration site, they need to be delivered in a matrix to allow
them to be released in a slow or sustained manner. A delivery in
saline or buffer alone is not effective in this regard. In this
example, a growth factor (bFGF) was used. bFGF (basic fibroblast
growth factor or FGF-2) is a growth factor known to stimulate
fibroblast proliferation and angiogenesis or blood vessel
formation. It was mixed with Aloe pectin (0.5%, w/v) in
physiological saline at a concentration of 1-10 .mu.g/ml and then
injected subcutaneously into the lower left or right side of
abdominal region of mice. One side received the control (pectin
alone), and the other side received the bFGF-containing
preparation. The in-situ gels from two mice were harvested along
with skin at days 5-10 and subjected to fixation in formalin,
sectioning, and H&E staining. Two identical areas, at either
end of the gel, vertically between the gel surface and the skin
muscle layer and horizontally 510 .mu.m inward from the lateral end
of the gel were selected, and the cells in these two selected areas
from each gel were numerated using the NIH image software. The
results showed that the cell number was more than 2 times higher in
bFGF-treated than the control (FIG. 5). An increase in blood vessel
formation surrounding the gel was also observed at a high bFGF
concentration (10 .mu.g/ml). This indicates that bFGF was released
from the in-situ gel and exerted its function in the surrounding
tissues.
EXAMPLE 13
In-Situ Gelation of a Dried Pectin Composition
A mixture of an Aloe pectin and CMC (0.75% by weight each) and 1.5%
CMC prepared in water were lyophilized in weighing trays,
separately. The dried materials were cut out as round pads (about 1
cm in diameter and about 3 mm in thickness) and were immersed in a
10 ml of normal calf serum in a petri dish. The Aloe pectin/CMC pad
formed a clear gel which remained intact for four days until the
experiments were terminated, whereas pads containing CMC alone were
dissolved or disappeared in a few hours under the same conditions.
Thus, these results show that pectin in a dried form can also form
a gel after being immersed in a body fluid.
EXAMPLE 14
Use of Pectin In-Situ Gel for Drug Delivery: Formulation
Process
The pectin in-situ gel can be used to provide a physiologically
acceptable composition that contains a therapeutic or diagnostic
agent and a low concentration of a gelling polymer (pectin) with a
pH and osmotic pressure characteristic of the body fluids, and that
has the capability to change from liquid to gel upon
administration.
The process to prepare a liquid formulation includes the following
steps.
1. Pectin is dissolved in sterile water.
2. A buffered or non-buffered saline is prepared.
3. The two solutions are mixed.
4. A physiologically active compound is added to the preparation at
step 3. The physiologically active agent may alternatively be added
to either solution before mixing.
Besides water and buffered or non-buffered saline or aqueous
solution, other pharmaceutically acceptable carriers may also be
used, including emulsions such as an oil/water emulsion, adjuvant,
various types of wetting agents, tablets, and capsules.
The pH of the formulation is adjusted with suitable buffering
agents such as boric acid-sodium borate, sodium phosphate
(monobasic)-sodium phosphate (dibasic), and Tris-HCl. Osmotic
pressure of the formulation is adjusted to mimic that of body
fluids with salts such as NaCl, KCL and MgCl.sub.2, and other
osmotic adjusting agents such as sorbitol, sucrose, glycerine, and
mannitol.
A pharmaceutically acceptable thickener may be added. The thickener
can be CMC, HPMC, HEC, alginate, gelatin, dextran, cyclodextrin, or
hyaluronic acid.
The formulation may be stored at room temperature or refrigerated
(4.degree. C.). If the formulation contains .about.0.15 M NaCl, a
(sodium) gel is formed when it is stored at 4.degree. C. Prior to
application, the gel is allowed to revert back to solution at room
temperature. For drug or therapeutic agents that are particulate,
prone to aggregate formation, or have a low water solubility such
as silvadene (silver sulfadiazine), storage in a gel matrix may be
advantageous because it may prevent aggregate or precipitate
formation.
Alternatively, the formulation may be prepared in a dried form. A
mixture of a pectin and a physiologically active agent in buffered
or non-buffered water or saline are lyophilized. Alternatively, a
pectin powder and a dry physiologically active agent are blended
and compressed into a desired form. The dried form may be used as a
pad, a tablet, a capsule, or a powder.
The relative amounts of the physiologically active agent and the
pectic substance in the formulation or composition can vary widely
dependent on the particular agent to be delivered. In a liquid
formulation, the agent can range from about 0.01% to about 50%
(w/v) while the pectic substance can range from about 0.01% to
about 40% (w/v). In a dried or suspended formulation, either the
agent or the pectic substance can range up to over 90% (w/w).
While the preferred compositions or formulations and methods have
been disclosed, it will be apparent to those skilled in the art
that numerous modifications and variations are possible in light of
the above teaching. It should also be realized by those skilled in
the art that such modifications and variations do not depart from
the spirit and scope of the invention as set forth in the appended
claims.
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